Carlile, Matthew
(2023)
APPLICATION OF NANOPORE DATA TO THE PIMMS (Pragmatic Insertional Mutation Mapping System) SEQ PIPELINE.
MRes thesis, University of Nottingham.
Abstract
Significant developments in next generation sequencing technologies have over the past 15 years allowed novel and exciting opportunities to further our understanding of genomic science. Transposons are mobile regions of DNA that occur frequently throughout the genome, the insertion of these transposable elements play an essential role in both gene regulation and evolution. Locating the positions of transposon insertion into a genome can help our understanding of the role they play in such areas as gene expression and structural variation. Random transposon mediated mutagenesis has proven to be a useful tool in the identification of essential and conditionally essential genes within various bacterial species. Various methods have been generated to identify the regions within a given genome where transposon insertion has occurred. Some of these methods involve complex experimental design in the laboratory, and a high degree of competency in bioinformatics post sequencing.
PIMMS (Pragmatic Insertion Mutation Mapping System) Seq was introduced in 2016 to speed up and simplify the bioinformatic pipeline when mapping transposon insertion sites using random mutagenesis sequencing data. The bacterial samples in question undergoing random mutagenesis, followed by inverse PCR, library preparation and sequencing (using Illumina technology). The use of Nanopore sequencing technologies has steadily increased since its introduction in 2014. Although the data quality is not yet as accurate as Illumina, Nanopore sequencing does offer some unique advantages, including the ability to sequence native DNA, generate long sequencing reads, and facilitate real time data analysis.
Oxford Nanopore Technologies (ONT), the company that developed nanopore sequencing also offer some unique sample enrichment options such as Cas-9 targeted sequencing which, for PIMMS could potentially be used as an alternative to inverse PCR, targeting sequences that contain known transposon motifs that flank a genomic region of interest.
Nanopore sequencing data was generated using S. agalactiae insertion mutant libraries, and uploaded to the PIMMS Seq bioinformatic pipeline to generate a database of transposon insertion sites. When comparing PIMMS output data generated using Nanopore sequencing to the established Illumina short read sequencing method, although the genomic distribution of insertions where consistent, differences where observed with Nanopore sequencing identifying 405 more unique insertion events, reducing the list of genes that were considered to be essential using short read sequencing. However, any advantages of this long read sequencing method must be off set against Nanopore’s sequencing base calling accuracy, which was significantly lower than that generated using Illumina technologies.
The Cas-9 Targeted library preparation from ONT has been proven to enrich for transposon sequences that flank genomic regions of interest, with on average 68.1% of all reads mapping to the insert sequence. This establishes its potential as an attractive alternative to amplification based methods. This method may significantly reduce the total amount of sequencing required to run an experiment, which in turn will reduce time and costs. Any potential wet lab sample loses are minimised due to a far smaller amount of sample processing steps.
Moving forward, nanopore long read sequencing is a technology that offers some unique advantages over short read technologies, and its application to the PIMMS pipeline has been demonstrated to work well. The added option of Cas- 9 Targeted nanopore sequencing, saves not only lab time and costs, but more importantly removes any potential amplification bias.
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